VENUS 74 (1–2): 1–17, 2016 New Species of Provanna from SW Japan ©Malacological Society of Japan1

Four New Species of Provanna (: Provannidae) from Vents and a Seep off Nansei-shoto Area, Southwestern Japan

Takenori Sasaki1*, Tomomi Ogura2,3, Hiromi Kayama Watanabe3 and Katsunori Fujikura2,3 1The University Museum, The University and Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 2Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan 3Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima-cho, Yokosuka-shi, Kanagawa 237-0061, Japan

Abstract: Members of the genus Provanna are abundant and widely distributed in deep-sea chemosynthetic environments. Around Japan provannids inhabit a wide range of depths and substrates. In this study, we report additional species of Provanna from vents and a seep in the Nansei-shoto area. Shell morphological and molecular phylogenetic analyses showed that there are four new species: (1) P. subglabra is characterized by an inated smooth shell and is most abundant in vent elds in the Okinawa Trough; (2) P. clathrata has a roughly sculptured shell and is distributed in vents in the southern Okinawa Trough; (3) P. lucida possesses a thin smooth shell with a deep suture and is limited to vents on the Minami-Ensei Knoll in the northern Okinawa Trough; and (4) P. kuroshimensis, which is distinguished by an olive-colored periostracum and is endemic to a seep site on the Kuroshima Knoll. In contrast to shell and molecular characters, radula morphology does not show clear differences among these species. The present study revealed a high species diversity of Provanna in a relatively limited area in southwestern Japan. The diversi cation of the genus might be correlated with complex topographic features in the Nansei-shoto area including the Okinawa Trough and Ryukyu Arc.

Keywords: Provannidae, Provanna, new species, Nansei-shoto area, Okinawa Trough, Japan

Introduction

The genus Provanna is one of the most abundant and widely distributed taxa in deep-sea chemosynthetic communities, including hydrothermal vents, hydrocarbon seeps, sunken wood and vertebrate falls. They are considered to be deposit feeders and grazers on bacterial mats and detritus (Sasaki et al., 2010). Molecular phylogenetic analyses have indicated that the Recent lineages of Provanna could have radiated about 15–45 million years ago (Johnson et al., 2010), and actual fossil records of Provanna trace back to the Cenomanian age in the Late epoch (Kaim et al., 2008). Up to now the genus has been composed of 25 valid species (see discussion for details) including 18 extant (Warén & Bouchet, 1986, 1993, 2009; Okutani, 1990; Warén & Ponder, 1991; Okutani, 1990; Okutani et al., 1992; Okutani & Fujikura, 2002) and seven fossil species (Squires, 1995; Kaim et al., 2008, 2009; Saether et al., 2010; Amano & Jenkins, 2013; Amano & Little, 2014). Among them only three living species have been known from the northwestern Paci c: (1) Provanna abyssalis has been collected from a methane seep site at a depth of 5,379 m on the

* Corresponding author: [email protected] DOI: http://doi.org/10.18941/venus.74.1-2_1 2 T. Sasaki et al. landward slope of the Japan Trench (Okutani & Fujikura, 2002); (2) P. shinkaiae was reported from another methane seep site at 5,343 m on the same landward slope (Okutani & Fujikura, 2002); (3) Provanna glabra inhabits a methane seep at depths around 1,100 m off Hatsushima Island, Sagami Bay (Okutani et al., 1992; Sasaki et al., 2007). A species of Provanna from the Okinawa Trough was previously identi ed as P. glabra, but it was later regarded as a separate species, based on differences in shell morphology (Sasaki et al., 2005, 2010). In addition, another similar species was collected from a seep on the Kuroshima Knoll which is the type locality of two species of Bathymodiolus (Okutani et al., 2004). Including these unnamed species, we describe four new species from Japan in this study.

Material and Methods

Study sites and sampling: Specimens of Provanna were collected from 10 sites around Japan between May 2002 and October 2011 during dives of the human-occupied vehicles (HOVs) Shinkai 2000 and Shinkai 6500 and the remotely operated vehicles (ROVs) Dolphin-3K, Hyper-Dolphin,

Fig. 1. Map of the sampling locations. a, whole map; b, magni ed map of the Nansei-shoto area. Circles, methane seep sites; triangles, hydrothermal vent sites. 1, Off Hatsushima Island site; 2, Minami-Ensei Knoll; 3, North Knoll of Iheya Ridge; 4, Iheya Ridge; 5, Izena Hole; 6, Irabu Knoll; 7, Hatoma Knoll; 8, Dai-yon Yonaguni Knoll; 9, Kuroshima Knoll. See Table 2 for geographic distribution of known Provanna species. New Species of Provanna from SW Japan 3

Table 1. List of sampling locations in this study. Number of sampling site corresponds to number in Fig. 1. No. Site Latitude Longitude Depth Habitat 1 Off Hatsushima Island, Sagami Bay 35°00′N 139°14′E 1,172 m Seep 2 Minami Ensei Knoll 28°24′N 127°38′E 701 m Vent 3 North Knoll of Iheya Ridge 27°48′N 126°54′E 982 m Vent 4 Iheya Ridge 27°33′N 126°58′E 1,399 m Vent 5-1 JADE site in Izena Hole 27°16′N 127°04′E 1,309 m Vent 5-2 HAKUREI site in Izena Hole 27°15′N 127°04′E 1,617 m Vent 6 Irabu Knoll 25°14′N 124°52′E 1,646 m Vent 7 Hatoma Knoll 24°52′N 123°51′E 1,473 m Vent 8 Dai-yon Yonaguni Knoll 24°51′N 122°42′E 1,387 m Vent 9 Kuroshima Knoll 24°08′N 124°12′E 644 m Seep and Kaiko 7000II of the Japan Agency for Marine-Earth Science and Technology (Fig. 1; Table 1). Live-collected specimens were xed and preserved in 99.5% ethanol or frozen. Morphological analyses: Species identi cation of Provanna has been based on shell and radular morphology (Warén & Ponder, 1991), and we also utilized these characters for morphological observations. We also performed morphometric analysis of the shell characters. Shell: Photographs of shells were taken using a digital camera system (ACT2-U) attached to a compound microscope system (MZ-3; Leica Microsystems). Seven shell morphology parameters, namely shell width (SW), height of body whorl (HBW), height of aperture (HA), width of aperture (WA), width of suture (WS), width of penultimate whorl (WP), and apical angle of shell (AS), were measured for all available spec imens in photos using Image J software (Abràmoff et al., 2004), which was installed in the ACT2-U system. Depth of suture (DSt) was calculated using the following equation: DSt = (WP – WS)/2. To compare the proportion of shells among different size samples, HBW and WA were divided by WA and SW, respectively, to minimize the effect of size variation. Additionally, measurements of the thickness of the peristome (TP), shape of aperture, and sculpture were obtained for species comparison. TP, DSt, and aperture were transformed into arbitrarily scored data based on whether it was thick or thin, sculptured or not sculptured, or pyriform or not pyriform in shape. The obtained morphometric data, HBW/SW, HA/WA, DSt, AS, and TP, were standardized to Euclidean distances. The standardized variables were transformed into a resemblance matrix, which was calculated using the Euclidean distances and used to perform cluster analysis using the SIMPROF test, with the level of signi cance set as α = 0.05. These statistical analyses were performed using PRIMER-E ver. 6 (Clarke & Gorley, 2006). Radula: Radulae were extracted from ethanol- xed specimens, cleaned in a diluted solution of commercial bleach, and mount ed on double-sided carbon tape (8 mm × 20 m; Nissin EM Corporation, Tokyo, Japan) under a compound microscope. The tape-mounted radulae were examined using a scanning electron microscope (TM3000; HITACHI Ltd., Tokyo, Japan). DNA extraction and ampli cation: Genomic DNA was extracted from the foot muscle, using the DNeasy Tissue Extraction Kit (QIAGEN). Each 1 μL genomic DNA was puri ed using GeneReleaser (BioVentures Inc., Marf reesboro, USA), following the manufacturer’s protocol. Two fragments of the mitochondrial cytochrome c oxidase subunit I (COI) were ampli ed by polymerase chain reaction (PCR) using the primer set LCO1490 and HCO2198 (Folmer et al., 1994), and newly designed primers, Pg501L (5′-TATACGATGACGGGGAATGC-3′) and Pg1253R (5′-TGTTGAGGAAAGAAAGTAATATTAA-3′) and combined into a single 1,044-bp sequence. In additi on, a 441-bp sequence of the 16S ribosomal RNA (16SrRNA) and a 434-bp sequence of the 4 T. Sasaki et al.

28S ribosomal RNA-D6 (28SrRNA-domain 6) were ampli ed using two primer sets, 16Sar and 16Sbr (Palumbi, 1996), and 28SD6F (McArthur & Koop, 1999) and 28SD6R (Colgan et al., 2003), respectively. PCR was conducted in a 20-μL solution that included 3 μL of puri ed template DNA, 11.3 μL of deionized sterilized water, 2 μL of 10 × PCR buffer, 1.5 μL of 2.5 mM dNTP, 1 μL of each primer and 0.2 μL of 5 U/μL Ex Taq DNA polymerase (TaKaRa Bio Inc., Ohtsu, Japan). PCR ampli cation of COI fragments was carried out using an initial denaturation step of 94°C (120 s), followed by 30 cycles consisting of a denaturation step at 94°C (30 s), an annealing step at 45°C (30 s), and an extension step at 72°C (30 s), and nally an extension at 72°C (40 s). DNA sequencing: The PCR products were puri ed using ExoSAP-IT® (USB®, Affymax , Santa Clara, USA). The puri ed PCR products were subjected to a cycle sequencing reaction using the BigDye® Terminator Cycle Sequencing Kit Version 3.1 (Applied Biosystems®, Life Technologies Corporation, Carlsbad, USA). The cycle sequencing reaction was conducted using 10 μL of a solution that included 1 μL of puri ed PCR products, 7.55 μL of deionized sterilized water, 0.7 μL of 5X BigDye Sequ encing Buffer, 0.25 μL of one of the primers used in PCR, and 0.5 μL of BigDye®, and was carried out under an initial denaturation step at 96°C (60 s), followed by 25 cycles consisting of a denaturation step at 96°C (10 s), an annealing step at 50°C (50 s), and an extension step at 60°C (60 s). The products were puri ed using the BigDye XTerminator® Kit (Applied Biosystems®) or Gel Filtration Cart ridge (Edge BioSystems, Gaithersburg, USA). The puri ed products were sequenced using an ABI 3130 automated DNA sequencer (Applied Biosystems®). The obtained sequence data were assembled into contigs by each locus using the program ATGC ver. 6.0.3 (Genetyx Corporation, Tokyo, Japan). Phylogenetic analyses: Molecular phylogenetic analyses were carried out to assess phylogenetic relationships of Provanna phylogroups collected from the Nansei-shoto area and Sagami Bay. Phylogeny of Provanna spp. inhabiting the East Paci c was analyzed with part of the dataset provided by Johnson et al. (2010). The sequence data of Abyssochrysos sp. and Desbruyeresia melanoides were used as outgroups. The sequences of mitochondrial COI (347 bp) and 16S rRNA genes (441 bp), and nuclear 28S rRNA gene (434 bp) were concatenated using Kakusan 4. Sequence alignment was performed using ProAlign ver. 0.5a3 (Löytynoja & Milinkovitch, 2003) after guide tree calculation with MAFFT version 6.925 (Katoh & Toh, 2008). The posterior probability scores were calculated using ProAlign, with scores of 40% and higher indicating that sites are ambiguous and should thus be eliminated from the analysis. Maximum-likelihood (ML) trees for molecular phylogenetic classi cation were constructed, using RAxML 7.2.8 Alpha (Stamatakis, 2006), which followed a General-Time-Reversible + Gamma (GTR + G) substitution model, according to the instruction of the manual. Robustness of nodes in the best scoring tree was assessed using the bootstrapping algorithm (1,000 replicates) (Efron, 1982). Appropriate evolutionary models for each gene were selected using the Bayesian Information Criteria (BIC) in Kaku san 4 (Tanabe, 2007) for the Bayesian analyses. Bayesian trees were also reconstructed from the sequences of the three loci using MrBayes ver. 3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) using the suitable substitution model estimated by BIC: JC69 + I + G for 28S, HKY85 + G for 16S, HKY85 + G for COI (1st), K80 + I for COI (2nd), and F81 + G for COI (3rd). This analysis inv olving four chains was run 3–4 × 106 generations with a printing frequency of 1,000, sampling frequency of 1,000, and burn-in of 30,000. Fig Tree ver. 1.3.1 (Rambaut, 2009) was used to display the generated ML trees. Abbreviations: The following abbreviations are used in the systematic descriptions. AS – apical angle of shell; DSt – depth of suture; HA – height of aperture; HBW – height of body whorl; JAMSTEC – Japan Agency for Marine-Earth Science and Technology; MO 1–4 – morphotypes 1–4; SL – shell length; SW – shell width; TP – thickness of peristome; UMUT – Department of Historical Geology and Paleontology, The University Museum, The University of Tokyo; WA – width of aperture; WP – width of penultimate whorl; WS – width of suture. New Species of Provanna from SW Japan 5

Table 2. List of described species of Provanna. † = fossil species. Species are arranged in alphabetical order. See also Sasaki et al. (2010: appendix 7.3) for extant species and Saether et al. (2010: table 2), Amano & Jenkins (2013: table 2) and Amano & Little (2014: table 1) for extinct species. Species Distirbution P. abyssalis Okutani & Fujikura, 2002 Japan Trench, 5,379 m, seep. P. admetoides Warén & Ponder, 1991 Florida Escarpment, 624–631 m, seep. †P. alexi Amano & Little, 2014 Shosanbetsu Village, northwestern Hokkaido, Japan, Chikubetsu Formation, Middle Miocene, whale-fall. †P. antiqua Squires, 1995 Washington State, USA, Eocene to Oligocene, seep and wood-fall. P. buccinoides Warén & Bouchet, 1993 Lau Basin and North Fiji Basin, 1,900–2,765 m, ve nt. P. chevalieri Warén & Bouchet, 2009 Regab site, West Africa, 3,150 m, seep. P. clathrata n. sp. Irabu Knoll and Hatoma Knoll, Okinawa Trough, Japan, 1,647–1,743 m, vent. P. glabra Okutani, Tsuchida & Fujikura, 1992 off Hatsushima, Sagami Bay, Japan, 1,110–1,200 m, seep. P. goniata Warén & Bouchet, 1986 Guaymans Basin, 2,000–2,020 m, seep. †P. hirokoae Amano & Little, 2014 Joetsu City, Niigata, Japan, Ogaya Formation, Middle Miocene, seep. P. ios Warén & Bouchet, 1986 East Paci c Rise 21°N–17°S, Galapagos Spreading Center, 2,450–2,620 m, vent. P. kuroshimensis n. sp. Kuroshima Knoll, Okinawa, Japan, 644 m, seep. P. laevis Warén & Ponder, 1991 Gulf of California, Guaymas Basin, Oregon Margin, Juan de Fuca Ridge, 500–2,000 m, vent and seep. P. lomana (Dall, 1918) Oregon Margin, ca. 450–1,200 m, seep. P. lucida n. sp. Minami-Ensei Knoll, Okinawa Trough, Japan, 701 m, vent. P. macleani Warén & Bouchet, 1989 Oregon Margin, 2,713–2,750 m, seep and sunken drift wood. †P. marshalli Saether, Little & Campbell, 2010 East Coast Basin, North Island, New Zealand, Bexhaven and Ihungia Limestone Formation, Early to Middle Miocene, seep. P. muricata Warén & Bouchet, 1986 East Paci c Rise 21°N, Galapagos Spreading Center, and North Fiji and Lau Back-Arc Basins, 2,450–2,615 m, vent. †P. nakagawaensis Kaim, Jenkins & Hikida, Nakagawa area, northwestern Hokkaido, Yezo Group, 2009 Coniacian and Campanian, seep and wood-fall. P. nassariaeformis Okutani, 1990 Mariana Back-Arc Basin, Manus Back-Arc Basins, 3,670–3,680 m, vent. P. reticulata Warén & Bouchet, 2009 Regab, Guiness and MPS 1-Congo sites, West Africa, 750–3,150 m, seep. P. sculpta Warén & Ponder, 1991 Louisiana Slope, ca. 550 m, seep. P. segonzaci Warén & Ponder, 1991 Lau Back-Arc Basin, 1,750–1,900 m, vent. P. shinkaiae Okutani & Fujikura, 2002 Japan Trench, 5,343 m, seep. P. subglabra n. sp. Okinawa Trough, Japan, 710–1,632 m, vent. †P. tappuensis Kaim, Jenkins & Waren, 2008 Tappu area, northwestern Hokkaido, Japan, Yezo Group, Cenomanian, seep. †P. urahoroensis Amano & Jenkins, 2013 Urahoro, eastern Hokkaido, Japan, Nuibetsu Formation, Oligocene, seep. P. variabilis Warén & Bouchet, 1986 Juan de Fuca Ridge, Endeavour Segment, Gorda Ridge, Oregon Margin, 675–2,200 m, vent, seep. 6 T. Sasaki et al.

Results

We recognized four new species as described below through analyses of molecular phylogeny, morphometrics, and conchological and radular morphology. Morphometric analyses: Cluster analysis identi ed four morphotypes (MO1 to MO4) in three major clusters in the dendrogram, viz. MO2, MO3, and a large cluster containing MO1 and MO4 (Fig. 2). This result indicates that MO1 ( = P. subglabra n. sp.), MO2 ( = P. clathrata n. sp.), MO3 ( = P. lucida n. sp.) are easily distinguished by a combination of proportion of height of body whorl and shell width (HBW/SW), proportion of height and width of aperture (HA/WA), depth of suture (DSt), apical angle (AS), and the thickness of peristome (TP). MO4 ( = P. kuroshimensis n. sp.) and similar specimens from Sagami Bay (P. glabra) could not be separated by a clear threshold in the characters used in the present study. However, these two lin eages were distinguished by molecular characters. In shell morphology, P. glabra and P. kuroshimensis is separable in curvature of columellar lip and color of periostracum. Phylogenetic analysis: The obtained sequences were registered to DDBJ, EMBL, and GenBank as Accession Numbers AB810040–AB810216 (COI), AB845559–AB845573 (16S), and AB845574–AB845588 (28S). Based on partial COI, 16S and 28S sequences, phylogenetic trees were reconstructed (Fig. 3). In addition to the already known P. glabra from Sagami Bay, Provanna collected from Nansei-shoto area were divided into four clades. These clades were completely congruent with the four morphotypes (MO1–4) revealed by the cluster analysis (Fig. 2). Therefore, we regard them as separate species and name MO1 P. subglabra n. sp., MO2 P. clathrata n. sp., MO3 P. lucida n. sp. and MO4 P. kuroshimensis n. sp., as described below. The clade of P. glabra is distinct from any species from the Nansei-shoto area but showed closer relationships with Provanna laevis in the Eastern Paci c (Fig. 3). The monophyly of the four new species were strongly supported by high bootstrap values (97–100%) and posterior probability (1 for all). Among the four new species, P. lucida n. sp. and P. kuroshimensis n. sp. form a clade, and P. subglabra n. sp. and P. clathrata n. sp. are united as another clade. Monophyly of each species was also supported robustly (Fig. 3) Shell characters: The four new species were de ned by shell sculpture, shell thickness, depth of suture, color of periostracum and curvature of columella (see diagnoses of species below). Opercula are indistinguishable among the four new species. Radular characters: Radulae of Provanna observed in this study were all similar and of taenioglossate type (Fig. 4). The rachidian tooth is triangular in shap e and possesses a single triangular central cusp and tripod-like bases. The lateral teeth have a slightly skewed triangular central cusp accompanied by a medium-sized inner cusp and approximately three smaller outer cusps diminishing in size distally. However, the number of inner cusps of a few specimens was variable intraspeci cally, particularly within P. subglabra n. sp. (Fig. 4F). This variability occurr ed in specimens within the same habitat. The marginal teeth bear a rake-like head. Radulae of the four newly described species did not show clear differences due to the variability of the lateral tooth (Fig. 4A–D). On the other hand, the radula of P. glabra showed a narrower radula ribbon and sharper rachidian tooth base compared to those of the four species.

Systematic Descriptions

Class Gastropoda Cuvier, 1795 Clade Caenogastrop oda Cox, 1960 Superfamily Abyssochrysoidea Tomlin, 1927 Family Provannidae Warén & Ponder, 1991 Genus Provanna Dall, 1918 New Species of Provanna from SW Japan 7

Fig. 2. Dendrogram based on clustering analysis on shell morphometry. Horizontal axis is the distance of similarity. Bold lines indicated a signi cant cluster (P < 0.05) by SIMPROF test. 8 T. Sasaki et al.

Fig. 3. Bayesian and maximum likelihood phylogenetic tree of the genus Provanna, using concatenated sequences of COI, 16SrRNA, 28SrRNA genes. Numbers beside branches represent posterior probability (left) and bootstrap values (right). Desbruyeresia melanoides and Abyssochrysos sp. are the outgroups. Abbreviations for localities: H, Hatoma Knoll; I, Irabu Knoll; K, Kuroshima Knoll; M, Minami-Ensei Knoll.

Provanna subglabra n. sp. (Fig. 5)

Zoobank ID: E6B6785A-85F7-4267-A442-C155F73139F1. Diagnosis: Adult shell smooth with swollen body whorl and curved columella. Juvenile shell with weak axial ribs, slightly angulate periphery, elevated spire, and straight columella. Suture not deep. Periostracum yellowish brown. Description: The adult shell is wholly smooth with the swollen body whorl and obviously curved columella. Juveniles possess more highly elevated upper whorls (Fig. 5E). The periostracum is bright yellowish brown in younger specimens and becomes thicker and deeper brown in color with growth. Initial whorls in juveniles have weak axial ribs and a slightly angulate periphery, but this ornamentation is gradually lost during ontogeny and disappears entirely in subadults and adults. The growth lines are indistinct in young specimens; rough lines are marked in the body whorls of adult specimens, especially at the last growth stage. The suture is not deeply formed throughout ontogeny. The columella is almost straight in juveniles and becomes more prominently curved later in ontogeny. The abapical end of the columella is a little twisted and forms a short notch. The upper whorls are often heavily corroded in gerontic specimens (Fig. 5A–C). The protoconch is missing in all specimens investigated. The operculum is typical for the genus. Etymology: The speci c name subglabra is given due to the similarity to P. glabra. Type locality: Hatoma Knoll, Okinawa Trough, Japan, 24°51.477′N, 122°50.507′E, 1,477 m, vent. Distribution: Hatoma Knoll, Izena Hole, Irabu Knoll and Minami Ensei Knoll in the Okinawa Trough, 710–1,632 m, all vents. Type specimens: Holotype, UMUT RM31137, SL = 5.5 mm, SW = 4.5 mm (Fig. 5A–C). New Species of Provanna from SW Japan 9

Fig. 4. Radula morphology of ve Japanese species of Provanna. A, P. subglabra n. sp., Hatoma Knoll; B, P. clathrata n. sp., Irabu Knoll; C, P. lucida n. sp., Minami-Ensei Knoll; D, P. kuroshimensis n. sp., Kuroshima Knoll; E, P. glabra, off Hatsushima; F, irregular form of the radula within P. subglabra n. sp. from Hatoma Knoll. Scale bars = 50 μm.

Paratype #1, UMUT RM 31138, SL = 6.0 mm, SW = 4.0 mm (Fig. 5D); paratype #2, UMUT RM 31139, SL = 5.2 mm, SW = 3.3 mm (Fig. 5E); paratype #3, UMUT RM31140, SL = 6.3 mm, SW = 4.6 mm (Fig. 5F); paratype #4, UMUT RM31141, SL = 6.0 mm, SW = 4.7 mm (Fig. 5G); paratype #5, UMUT RM31142, SL = 5.7 mm, SW = 4.0 mm (Fig. 5H); paratype #6, UMUT RM31143, SL = 3.7 mm, SW = 2.5 mm (Fig. 5I). Remaining paratypes used for morphometric analysis and DNA sequencing were deposited in JAMSTEC. Holotype and paratypes #1, 2 from the type locality. Paratype #3 from Hakurei Site, Izena Hole, Okinawa Trough, Japan, 27°14.815′N, 127°04.089′E, 1,617 m. Paratypes #4–6 from Irabu Knoll, Okinawa Trough, Japan, 25°13.768′N, 124°52.172′E, 1,632 m. Remarks: This species was listed as Provanna sp. by Sasaki et al. (2010). It had been previously identi ed as Provanna glabra (Okutani et al., 1993; Okutani & Fujiwara, 2000), but now can be distinguished by juvenile sculpture and columellar curvature (Sasaki et al., 2005: 118; Sasaki et al., 2010: 239). Provanna glabra is endemic to seep sites off Hatsushima, Sagami Bay, Japan. Recently, the whole mitochondrial genome was sequenced for an unidenti ed species of the genus 10 T. Sasaki et al. by Xu et al. (2015). From shell morphology and COI sequences, their Provanna sp. seems to be identi able as this new species, although it was collected from a seep. If that is the case, geographic distribution of this species is extended to the South China Sea.

Fig. 5. Provanna subglabra n. sp. A–C, holotype, UMUT RM31137; D, paratype #1, UMUT RM 31138; E, paratype #2, UMUT RM 31139; F, paratype #3, UMUT RM31140; G, paratype #4, UMUT RM31141; H, paratype #5, UMUT RM31142; I, paratype #6, UMUT RM31143. A–E, Hatoma Knoll; F, Hakurei Site, Izena Hole; G–I, Irabu Knoll. New Species of Provanna from SW Japan 11

Provanna lucida n. sp. (Fig. 6)

Zoobank ID: B76C21A8-47CC-41B7-9918-180487B3EA61. Diagnosis: Shell thin, smooth and polished with deep suture. visible through shell. Adult shell with inated whorls; juvenile shell with slightly angulate periphery. Columella straight to C-shaped. Periostracum tan and very thin. Descriptions: The shell is thin, and the presence of the animal is discernible through the shell. The surface is smooth and polished with a very thin yellowish periostracum. Only in juveniles (Fig. 6D, E), the whorls are slightly angulate with a few indistinct spirals which are gradually lost with growth. The suture of whorls is deeply incised. The growth lines are hardly visible even in fully grown specimens. The curvature of the columella is variable: nearly straight in some specimens (Fig. 6B), but C-shaped in others (Fig. 6A, C). The protoconch is missing in all specimens

Fig. 6. Provanna lucida. A, holotype, UMUT RM31132; B, paratype #1, UMUT RM31133; C, paratype #2, UMUT RM31134; D, paratype #3, UMUT RM31135; E, paratype #4, UMUT RM31136. A–E, Minami-Ensei Knoll. 12 T. Sasaki et al. investigated. Etymology: The speci c name lucida is a Latin adjective meaning being clear, bright and shining. Type locality: Minami-Ensei Knoll, Okinawa Trough, Japan, 28°23.476′N, 127°38.392′E, 701 m, vent. Type specimens: Holotype, UMUT RM31132, SL = 5.9 mm, SW = 3.8 mm (Fig. 6A). Paratype #1, UMUT RM31133, SL = 6.0 mm, SW = 3.7 mm (Fig. 6B); paratype #2, UMUT RM31134, SL = 5.5 mm, SW = 4.1 mm (Fig. 6C); paratype #3, UMUT RM31135, SL = 4.3 mm, SW = 2.9 mm (Fig. 6D); paratype #4, UMUT RM31136, SL = 3.3 mm, SW = 2.3 mm (Fig. 6E). Remaining paratypes used for morphometric analysis and DNA sequencing were deposited in JAMSTEC. All from the type locality. Remarks: This species is the thinnest-shelled Provanna, although we could not obtain accurate thickness measurements for fragile type specimens. In other known species of the genus, the animal is not visible through the shell. Provanna abyssalis is similar to this species in having a deep suture of whorls. However, a very shallow siphonal notch is formed in P. lucida n. sp. but not in P. abyssalis.

Provanna clathrata n. sp. (Fig. 7A–B)

Zoobank ID: D22A110F-F178-4703-8C3D-9235406535F6. Diagnosis: Shell with cancellate sculpture, well swollen whorls and distinct suture. Columella twisted sigmoidally. Periostracum thick and yellowish brown. Description: The shell is roughly scul ptured and covered with a thick yellowish brown periostracum sometimes bearing rusty deposits. The whorls are highly elevated and moderately inated for the genus. The exterior sculpture above the periphery of the body whorl is cancellate and consists of three spiral ribs and constantly spaced axial ribs. Their intersections protrude as nodules. The periphery is marked by a single spiral rib which lacks nodes. The basal area below the periphery possesses three lesser spiral ribs. The columella is prominently twisted in a sigmoidal form at the abapical terminal. The protoconch is missing in all specimens investigated. Etymology: The speci c name clathrata indicates the distinctive latticed sculpture. Type locality: Irabu Knoll, Okinawa, Trough, Japan, 25°13.758′N, 124°52.204′E, 1,647 m, vent. Distribution: Irabu Knoll and Hatoma Knoll, Okinawa Trough, Japan, 1,647–1,743 m, all vents. Type specimens: Holotype, UMUT RM31148, SL = 5.4 mm, SW = 4.8 mm (Fig. 7A). Paratype #1, UMUT RM31149, SL = 5.7 mm, SW = 4.1 mm (un gured); paratype #2, UMUT RM31150, SL = 4.2 mm, SW = 2.8 mm (Fig. 7B). All from the type locality. Remaining paratypes from the Irabu and the Hatoma Knoll were used for morphometric analysis and DNA sequencing and deposited in JAMSTEC. Remarks: This new species is most similar to P. segonzaci (Warén & Ponder, 1991: g. 20G), but the tubercles at the intersections of the axial and spiral ribs are less prominent in P. clathrata n. sp.

Provanna kuroshimensis n. sp. (Fig. 7C–F)

Zoobank ID: BE607851-4094-44A2-BEB0-7284045568C3. Diagnosis: Shell smooth with olivaceous periostracum, lacking yellowish hue. In adult, whorls moderately inated with strongly curved columella. In juvenile, whorls more angula te at periphery with straight columella. Suture shallow both in adults and juveniles. Descriptions: The exterior surface is entirely smooth and covered with an olive-colored New Species of Provanna from SW Japan 13 periostracum. The whorls are scarcely inated in juveniles but swell obviously in fully grown adults. The growth lines are smooth in most areas, but rough lines are noticeable on the last part of the body whorl. The columella is nearly straight in juveniles but modi ed to be more obviously curved in adults. The abapical end of the columella is twisted but does not form a notch at the aperture. The suture is shallow throughout ontogeny. The protoconch is missing in all specimens investigated. Etymology: The speci c name represents the endemism of the species in a methane seep site in Kuroshima Knoll. Type locality: Kuroshima Knoll, southern Ryukyu Islands, Okinawa, Japan, 24°07.998′N, 124°11.502′E, 644 m, seep. Type specimens: Holotype, UMUT RM31144, SL = 5.2 mm, SW = 3.6 mm (Fig. 7C). Paratype #1, UMUT RM31145, SL = 5.3 mm, SW = 3.5 mm (Fig. 7D); paratype #2, UMUT RM31146, SL = 4.7 mm, SW = 3.3 mm (Fig. 7E); paratype #3, UMUT RM31147, SL = 2.5 mm, SW = 1.9 mm (Fig. 7F). Remaining paratypes used for morphometric analysis and DNA sequencing were deposited in JAMSTEC. All from the type locality.

Fig. 7. A–B. Provanna clathrata; A, holotype, UMUT RM31148; B, paratype #2, UMUT RM31150. C–F. Provanna kuroshimensis; C, holotype, UMUT RM31144; D, paratype #1, UMUT RM31145; E, paratype #2, UMUT RM31146; F, paratype #3, UMUT RM31147. A–B, Irabu Knoll; C–F, Kuroshima Knoll. 14 T. Sasaki et al.

Remarks: This species is most similar to P. subglabra n. sp. but distinguishable by the curvature of the columella and color of the periostracum. Geographic distributions are also separate between the two species.

Discussion

With the description of the four new species, the genus Provanna consists currently of 29 valid species (Table 2) including 22 extant (Warén & Bouchet, 1986, 1993, 2009; Okutani, 1990; Warén & Ponder, 1991; Okutani, 1990; Okutani et al., 1992; Okutani & Fujikura, 2002; this study) and seven fossil species (Squires, 1995; Kaim et al., 2008, 2009; Saether et al., 2010; Amano & Jenkins, 2013; Amano & Little, 2014). The present research using morphological and genetic analyses showed a high species diversity of Provanna in deep-sea chemosynthetic environments in the Nansei-shoto area: P. subglabra n. sp., P. clathrata n. sp. and P. lucida n. sp. are from vents in the Okinawa Trough, and P. kuroshimensis n. sp. inhabits a seep in the Kuroshima Knoll. Shell characters: The results of morphological analyses of shell characters showed that the four species in the Nansei-shoto area could be distinguished by ve characters. (1) Sculpture of shell surface: The shell surface is rough with axial and spiral ribs in P. clathrata n. sp. but smooth in the rest of the Japanese species. Sculptural differ ence is notable in certain other species of Provanna (e.g., P. admetoides, P. sculpta and P. segonzaci: Warén & Ponder, 1991). (2) Color of periostracum: The periostracum is olive-colored in P. kuroshimensis n. sp., but it is more yellowish in the remaining species. This character is particularly useful to distinguish similar-looking P. kuroshimensis n. sp. and P. subglabra n. sp. Such a color difference is not known among other similar species in Provanna. (3) Depth of suture: Among smooth-shelled species, P. lucida n. sp. could be distinguished from the other two by a deep suture in whorls. (4) Shell thickness: In contrast to other species the shell of P. lucida n. sp. is so thin that the animal inside is visible through the shell. (5) Aperture form: The ratio of the height and width of the aperture is different among species: The aperture is most rounded in P. lucida n. sp., and more pointed anteriorly in P. clathrata n. sp., P. subglabra n. sp. and P. kuroshimensis n. sp. This difference is produced by a combination of the characteristics of whorl swelling and curvature of the columellar lip. Radula: The radula morphology did not show clear-cut differences among the four new species (Fig. 4A–D). Molluscan radulae are widely accepted as conservative characters but not always diagnostic at the species level. It is important to estimate the degree of morphological variation within a single locality, among different localities and also among different size classes to use this character for phylogenetic/taxonomic purposes. Phylogeny: Monophyly of each species was strongly supported by our molecular phylogenetic analysis (Fig. 3). In overall appearance of the shells, P. subglabra n. sp. (MO1) and P. kuroshimensis n. sp. (MO4) are similar as is shown in the morphometric analysis (Fig. 2). However, our molecular phylogenetic tree (Fig. 3) does not show such a relationship, and instead species pairs are formed between P. subglabra n. sp. and P. clathrata n. sp. and between P. lucida n. sp. and P. kuroshimensis n. sp. Interestingly it was revealed that P. laevis from Monterey Bay, California is nested within P. glabra from Sagami Bay (Fig. 3). This closeness might be regarded as unlikely given the geographic distance between the two localities. However, a similar relationship has been reported in Calyptogena inhabiting deep-sea chemosynthetic environments: Calyptogena soyoae Okutani, 1957 in Sagami Bay and C. kilmeri Bernard, 1974 in the Oregon Subduction Zone and Guaymas Transform Fault belong to the same clade (Peek et al., 1997; Kojima et al., 2004). Therefore, a larger-scale analysis of Provanna species between the East and West Paci c chemosynthetic sites New Species of Provanna from SW Japan 15 is necessary to clarify the relationship in detail. Habitat: Habitats of the new species described here are vents (P. subglabra n. sp., P. lucida n. sp., and P. clathrata n. sp.) and a seep (P. kuroshimensis n. sp.), and none of these species was found in both environments. This is also the case for other species of Provanna. Almost all species of the genus have been discovered only from a single type of environment (e.g., hydrothermal vents, hydrocarbon seeps, whale falls or sunken wood) and within a limited depth range: The exceptions are Provanna laevis and P. variabilis which have been recorded from both vents and seeps (see Sasaki et al., 2010: appendix 7.3). However, the past records of these two species might contain misidenti cation (Warén, personal communication). Geographic distribution: Distributions of the new Provanna species are restricted to rather small areas, except for P. subglabra n. sp., which is found from the Minami-Ensei Knoll (locality 2 in Fig. 1) to the Hatoma Knoll (locality 7 in Fig. 1) in the Okinawa Trough. If Provanna sp. in Xu et al. (2015) belongs to the latter species, its range would be greatly extended to the South China Sea. In the remaining species, P. clathrata n. sp., P. lucida n. sp. and P. kuroshimensis n. sp., have so far been collected only from the Irabu and Hatoma Knolls, Minami-Ensei Knoll and Kuroshima Knoll in the Okinawa Trough, respectively. Hence, it can be assumed that these Provanna species have different dispersal abilities. However, without data on reproduction, development, and genetic population structure, we cannot draw conclusions about the correlation between geographic distribution and life-history traits at present. In the Okinawa Trough, many organisms are known to be endemic to vent elds, and their populations are subdivided by the complex topography of the Ryukyu Arc (Watanabe et al., 2010). Speciation by geographic isolation might be common for vent- and seep-associated organisms in an arc-backarc system in the Nansei-shoto area.

Acknowledgements

We are grateful to the scientists and students of cruise NT11–20, the of cers and crew of the R/V Natsushima, and the operation team of the ROV Hyper-Dolphin. We also thank Drs. T. Okutani, C. Chen, T. Haga, J. Lorion, and R. Nakajima in JAMSTEC for their technical advice and useful discussion. The new name Provanna subglabra proposed here is originated from a personal idea by Dr. Takashi Okutani. This study was partially supported by the Trans-crustal Advection and In situ reaction of Global sub-seaoor Aquifer (TAIGA) project of the Ministry of Education, Culture, Sports, Science and Tech nology (MEXT), Grant-in- Aid for Scienti c Research B (26291077, 23370040, 15H04412) from Japan Society for the Promotion of Science, and the Environment Research & Technology Development Fund (S-9-5-6) of the Ministry of the Environment, Japan.

References

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(Received May 20, 2015 / Accepted February 25, 2016) New Species of Provanna from SW Japan 17

南西諸島沖の熱水噴出域および冷水湧水域から発見されたハイカブリナ属の 4 新種

佐々木猛智・小倉知美・渡部裕美・藤倉克則

要 約

Provanna Dall, 1918 ハイカブリニナ属は深海の化学合成生物群集に広く生息し多産するグループである。 日本の近海では，ハイカブリニナ属には P. glabra Okutani, Tsuchida & Fujikura, 1992 サガミハイカブリニ ナ，P. abyssalis Okutani & Fujikura, 2002 カイコウハイカブリニナ，P. shinkaiae Okutani & Fujikura, 2002 シ ンカイハイカブリニナの 3 既知種が分布しており，種によって深度分布と底質が異なる。本研究では南西 諸島海域から新たに発見された新種について報告する。貝殻の比較形態学的解析および分子系統解析の結 果から，4 新種の存在が明らかになった。（1）P. subglabra n. sp. ニヨリハイカブリニナ（似寄灰被蜷：和 名新称）は膨らみのある平滑な貝殻で特徴づけられ，沖縄トラフの熱水噴出域では最も多産する。種小名 と和名は，本種がかつて P. glabra サガミハイカブリニナに同定されていたことに由来する。（2） P. clathrata n. sp. コウシハイカブリニナ（格子灰被蜷：和名新称）は粗い格子状の彫刻を持ち，沖縄トラフ南部の熱 水噴出域に生息する。（3） P. lucida n. sp. ミガキハイカブリニナ（磨灰被蜷：和名新称）の殻は平滑で縫 合が深く，今のところ沖縄トラフ北部の南奄西海丘にのみ出現する。（4）P. kuroshimensis n. sp. クロシマ ハイカブリニナ（黒島灰被蜷：和名新称）の殻は平滑でオリーブ色の殻皮を持ち，黒島海丘に固有であ る。一方，歯舌の形態は 4 種の間で明確な差は見られなかった。この発見により，ハイカブリニナ属は南 西諸島の狭い範囲で多様化していることが明らかになったが，その要因としては南西諸島海域の化学合成 生態系形成域が多様な環境にあることが関係していると考えられる。